Assessment of the ecotoxicity of bismuth at the phytotoxicity of soils

. The results of the study of the ecotoxicity of bismuth on ordinary chernozem, brown forest soils and sierosands along the length of radish roots are presented. Small doses of 1.5-3 mg/kg of bismuth stimulated the growth of radish roots on ordinary chernozem. The maximum toxicity of bismuth carbonate and nitrate at a dose of 300 mg / kg was established on sierosands (reduction in the length of radish roots by 43% of the control). Bismuth carbonate 300 mg/kg showed the greatest toxicity when applied to ordinary chernozem and brown forest soil and reduced the length of radish roots by 31 and 44% of control, respectively. The series of toxicity ( (on radish’s root length) of chemical forms of bismuth for soils forms the following sequence: bismuth carbonate (84) ≥ bismuth nitrate (86) > bismuth oxide (90). The toxic effect of bismuth depends on the form and concentration of bismuth in the soil,the particle-size composition, the reaction of the soil environment and the content of organic matter in the soil.

higher plants. The need for a definition of this indicator occurs when the monitoring of chemical contamination of soils. One of the mechanisms of phytotoxicity of heavy metals is the formation of excess reactive oxygen species, which leads to oxidative stress in plant cells [11]. Another mechanism of phytotoxicity is the substitution of essential elements (Cd-Zn, Se-S) or the binding of heavy metals by enzymes, which leads to a violation of their function. In large quantities, essential chemical elements are toxic to plants and disrupt the functions of enzymes. As a result, plants synthesize more toxic thiol compounds [12]. Bismuth is not an essential element. However, it was previously established that bismuth can affect the transport of iron in plant cells [10]. Since the effects of bismuth on plants and soils are currently poorly understood, this determined the relevance of this study.
The aim of the study is to assess the ecotoxicity of bismuth by the phytotoxicity of soils.  [13] as Haplic Chernozems(Loamic) is characterized by a heavy loam the particle-size composition, an average organic matter content -3.7% and a reaction of soil pH=7.8 (slightly alkaline). Sierosands or Haplic Arenosols (Eutric) are characterized by light particle-size composition; low organic matter content-2.3% and reaction of soilpH, 6.8 (neutral). Brown forest soils or Haplic Cambisols (Eutric) are characterized by heavy loam the particle-size compositionloworganic matter content-1.8%; and reaction of soil рН=5.8 (acid).

Experimental details
Soil samples were taken from the upper soil layer (0-10 cm) because heavy metals are retained in it [4]. The soil samples differed in the types of land: arable land (ordinary chernozem), grass-grass steppe (sierosands) and beech-hornbeam forest (brown forest soil). The background content of bismuth in the objects of the study is 0.27, 0.28 and 0.14 mg/kg, respectively, in ordinary chernozem, brown forest soil and sierosands. The bismuth content was determined by inductively coupled plasma mass spectrometry (ICP-MS) at the All-Russian Geological Research Institute named after A. P. Karpinsky (St. Petersburg). The analysis of the literature data did not reveal the forms in which bismuth compounds enter the soil. Probably, it can be oxides, as well as soluble and insoluble salts. These forms of compounds are typical for most heavy metals that pollute the soil [4].The study evaluated the phytotoxic properties of soils, since they are the first to react to chemical contamination. The phytotoxic properties of the soils were evaluated after 10 days of contamination. A longer incubation period increases the differences in the state of the soil incubated in the laboratory from its state in natural conditions. The simulation of the experiment on soil contamination was carried outin triplicateunder laboratory conditions using the methods generally accepted in soil biology and ecology [14]. Bismuth (III) compounds were studied, since the trivalent state of bismuth is the most stable in nature [15]. According to authors [6][7][8][9][10][11], it was found that contaminated soils can contain bismuth in concentrations ranging from 1.5 mg / kg till 436 mg / kg and more then. The soils were polluted with bismuth oxide Bi2O3, bismuth carbonate (BiO)2CO3, and bismuth nitrate Bi (NO3)3in different concentrations -1.5, 3, 15, 30, 150 and 300 mg/kg. Taking into account the solubility of the compounds, the application of bismuth oxide, carbonate, and nitrate to the soil differed. Bismuth nitrate was dissolved in water and introduced into the soil at the first watering. Bismuth oxide and carbonate were ground with a small amount of dry soil. Then the soil was thoroughly mixed with the rest of the soil of the incubation vessel, after which watering was carried out. The soil (0.5 kg) was incubated at optimal humidity (60% of the field moisture capacity) and a temperature of 20-22°C in triplicated.
Sustainability in the right sense was evaluated by the length of the radish roots in relation to different doses of bismuth. According to [16], roots are more sensitive to heavy metal contamination by plant organs than shoots. Based on this, the phytotoxicity of soils was calculated by changing the length of the roots of radish (Raphanussativus L.). The change in the length of radish roots in the studied soils was expressed as a percentage relative to the control. The selected seeds were placed evenly in 5 rows of 5 seeds in Petri dishes with soil. Each variant of each soil type and form of chemical compound was studied in triplicate with subsequent statistical processing (n=225: 3 vegetative vessels in 3 biologicalreplicatesin Petri dishes× 25 radish seeds). The control was non-polluted soil with bismuth, selected from the studied areas.

Statistical analyses
To check the reliability of the results, an analysis of variance was carried out followed by the determination of the least significant difference (LSD). Variation statistics (mean values, dispersion) was determined, reliability of different samples was established by using dispersion analysis (Student-t) and the correlation analysis (Pearson correlation coefficient) was conducted.

Changes the radish's root length of chernozem ordinary upon contamination with bismuth
With the introduction of small doses of all chemical forms of bismuth (1.5 and 3 mg/kg) in ordinary chernozem, statistically unreliable stimulation of the radish root system was recorded (Fig.1). The stimulating effect of bismuth nanoparticles at a dose of 2.8 and 5.6 mg / kg on seed germination of spring wheat by 10 and 30% was previously established [17]. In the course of the experiment, the length of radish roots decreased in inverse proportion to the applied doses of all chemical compounds of bismuth to the soil by 15-31%of control.With the introduction of bismuth oxide in doses of 150 and 300 mg/kg, the root length indices decreased by 15 and 22% of control. The introduction of bismuth carbonate in doses of 30-300 mg/kg inhibited the radish root system by 18-31% of control. Bismuth nitrate, when applied at 30-300 mg/kg, showed a toxic effect and reduced the length of radish roots by 16-23% of control.
T. Nagata (2015) obtained similar results when studying the effect of bismuth nitrate on Arabidopsis thaliana. Small doses of bismuth 0.1 and 1 μM did not affect the growth of the plant root system and even stimulated root growth by 10-20% [10]. With an increase in the concentration of bismuth to 2μM, the length of Arabidopsis thaliana roots decreased by 50% from the control values. Bismuth nitrate at concentrations of 2 and 3 μM inhibited plant root growth and increased lateral root growth. The highest bismuth concentration of 6μM significantly reduced the root growth of Arabidopsis thaliana.

Changes the radish's root length of brown forest soil upon contamination with bismuth
When low concentrations of 1.5 and 3 mg/kg of nitrate and bismuth oxide were introduced into brown forest soil, no statistically significant differences from the control were observed (Fig. 2). A statistically significant decrease in the length of radish roots was recorded when 15 mg/kg of oxide, carbonate and bismuth nitrate by 13, 14 and 15% of control, respectively. When a dose of 30 mg / kg of bismuth oxide, carbonate, and nitrate were introduced into brown forest soil, the root length decreased by 15, 29, and 18% of control. Concentrations of bismuth oxide 150 and 300 mg/kg and bismuth nitrate at a dose of 150 mg/kg equally inhibited the length of plant roots by 26% of control. The introduction of a dose of 30-300 mg/kg of bismuth carbonate and nitrate reduced the growth of the radish root system by 18-42% of control.

Changes the radish's root length of sierosands upon contamination with bismuth
When sierosands were contaminated with low doses of bismuth (1.5 and 3 mg/kg) of all chemical forms, as well as when applied to brown forest soil, no statistically significant differences in the length of radish roots from the control values were found (Fig. 3). added to sierosands, bismuth nitrate showed slightly greater toxicity than the same doses of bismuth oxide and carbonate. Bismuth carbonate and nitrate, when added to sierosands 300 mg/kg, equally inhibited the radish root system by 43%. With an increase in the concentration of bismuth nitrate to 4 and 5 μM in Arabidopsis thaliana, the absence of the formation of normal tissues of the root system was observed [12].

Comparative assessment of the toxicity of bismuth compounds by the length of radish roots grown on different soils
An assessment was made of the ecotoxicity of bismuth compounds and the rsustainabilityof soils to contamination with bismuth along the length of the radish roots. Based on the chemical form of the pollutant, the averaged range of toxicity of bismuth for soils along the length of the radish roots is: bismuth carbonate (84) ≥ bismuth nitrate (86)> bismuth oxide (90). The biological effect of the effect of bismuth on plants depends not only on the dose, but also on the form of being in soils [9]. Bismuth oxide, as a less soluble substance in the soil, had a less toxic effect. Previously, the highest toxicity of nitrate ions was established when studying the biological properties of soils in Southern Russia [18]. In the present study, bismuth carbonate is the most toxic substance. This fact requires further deeper study.
Similar results in assessing the phytotoxicity of silver were obtained by N.I. . The authors observed a decrease in the length of the roots of radish grown on ordinary chernozem, sierosands and brown forest soil by 17, 24, and 29% of control, respectively, when the maximum dose of silver was applied to 100 mg/kg [19].
With an increase in soil pH, bound fractions of heavy metals increase due to the formation of a large number of pH-dependent cation-exchange sites on the soil surface and an increase in the stability constants of metal complexes with fulvic acids and humic acids [20]. A. KabataPendiasand X. Pendias(2010) established the formation of stable carbonates by bismuth in neutral soil [4]. Bismuth carbonates are not stable in acidic soils, compared to neutral and alkaline soils [21]. Carboxyl groups play a major role in the solubility of Bi in acidic and neutral media, while phenolic-OH, amino groups, and thiol groups (-SH) play an important role in neutral and alkaline media [22].The presence of a large amount of easily decomposed organic matter in the upper layers in the acidic brown forest soil contributes to the formation of a large number of soluble complexes of bismuth with organic matter [23]. Soil acidification increases the mobility and availability of many elements, including bismuth [24]. Thus, bismuth in ordinary chernozem exhibits the lowest ecotoxicity (on radish's root length) in comparison with other types of soils.

Conclusion
As a result of a study of the ecotoxicity of bismuth for phytotoxicity of different types of soils, a decrease in the length of radish roots was established. Low concentrations of 1.5-3 mg/kg of all chemical forms of bismuth had a stimulating effect on the length of the roots of radish on ordinary chernozem. The highest toxicity of bismuth carbonate and nitrate at a concentration of 300 mg/kg was found on sierosands (reduction in the length of radish roots by 43% from control). On ordinary chernozem and brown forest soil, the highest toxicity was foundwith the introduction of 300 mg/kg of bismuth carbonate -31 and 44% of control, respectively. The series of toxicity of chemical forms of bismuth for soils (on radish's root length) forms the following sequence: bismuth carbonate (84) ≥ bismuth nitrate (86)> bismuth oxide (90). The toxic effect of bismuth depends on the form and concentration of the element in the soil, the particle-size composition, the reaction of the soil environment and the content of organic matter in the soil. The greatest rsustainability to bismuth contamination of radishes grown on ordinary chernozem is due tothe particle-size compositionof the soil, the high content of organic matter (3.7%) and the slightly alkaline reaction of the soil (pH = 7.8). Bismuth is mobile in brown forest soil with an acidic reaction of the soil environment (pH = 5.8), and has a toxic effect on the root system of radish. The low toxicity of bismuth (on radish's root length) in the sierosands was established. The light particle-size composition of sierosands does not provide sufficient absorption capacity to fix bismuth in the soil, which requires further research.